Detecting and/or measuring a substance based on a resonance shift of photons orbiting within a microsphere

ABSTRACT

Detecting and/or measuring a substance based on a resonance shift of photons orbiting within a microsphere of a sensor. Since the resonance of the microsphere has a large quality factor, the sensor is extremely sensitive. The sensor includes the microsphere coupled with at least one optical fiber. The surface of the microsphere includes receptors complementary to the substance. The at least one optical fiber can be provided with at least one additional microsphere having a surface free of the receptors. Resonance shifts observed in such an additional microsphere(s) can be attributed to factors unrelated to the presence of the substance. The resonance shift observed in the microsphere with the receptors can be compensated based on the resonance shift of the additional microsphere(s) to remove the influence of these other factors.

§ 1. BACKGROUND

§ 1.1 Field of the Invention

The present invention concerns detecting the presence of, and/ormeasuring the amount or concentration of substances, such as chemicaland/or biological substances for example. More specifically, the presentinvention concerns detecting and/or measuring a substance based on aresonance shift of photons orbiting within a microsphere.

§ 1.2 Related Art—Measurement Principle Using Ray Optics In MicroscopicHaving a Changing Size

Resonances in a geometrical optics limit are associated with the opticalray paths, such as those 110 illustrated in the cross section of aparticle 100 illustrated in FIG. 1. Total internal reflection keeps thephoton(s) from radiating outward. Collectively, the ray path segments110 define a polygon.

Basically, the light circles (or orbits) the interior of the particle100, returning in phase. This is known as a mode of the first order. Forhigher order modes, the photon(s) takes several orbits before its raypath closes—i.e., before the photon returns in phase.

The foregoing illustration and assumptions are appropriate formeso-optic elements (i.e., devices, comparable in size to the wavelengthof light, that can confine photons) 100 having a diameter 2 a that isbetween 10 and 100 times the wavelength of the photon. The resonanceshave specific polarization states.

Referring to FIG. 2, an optical fiber 200 may be evanescently coupledwith a microsphere 100′. More specifically, an evanescentelectromagnetic field associated with total internal reflection existsjust outside the microsphere 100′, decaying exponentially as a functionof distance, typically over a distance of ˜0.1 μm. Further, internalreflection on a curved surface induces a small amount of radiationleakage in the far field. The higher the order of the mode, the greaterthe leakage. For example, the energy loss in one oscillation withinslightly spheroidal fused silica microspheres (2 a>˜50 μm) has beenmeasured to be smaller than 2 billionths of the energy contained,yielding a quality factor Q>˜10⁸. Stated in another way, the linewidthof the associated resonance (δf) in the spectrum is 10 billionth of thefrequency (δf=f/Q). Referring to FIG. 3, the resonance modes can bedetected as transmission dips 300 in the evanescently coupled opticalfiber 200.

As illustrated in FIG. 4, if the size (or shape, or refractive index) ofthe particle 100/100′ changes, the resonances shift in frequency. Forexample, in the case of a sphere, as its radius increases, the resonanceoccurs at a longer wavelength. This shift can be expressed as:

$\begin{matrix}{\frac{\Delta\; a}{a} = \frac{\Delta\;\lambda}{\lambda}} & (1)\end{matrix}$This relationship may be derived as follows.

When considering size sensitivity, recognize that the angular momentum Lof the photon in a given mode is quantized. That is

${L = {( \frac{h}{2\;\pi} )\sqrt{l( {l + 1} )}}},$where l is an integer and h is Plank's constant. The angular momentum inthe geometry of FIG. 1 is equal to its linear momentum (p) times thedistance of the closest approach from the sphere center (a cos(π/q)),where q is the number of reflections in the orbit. The linear momentum pof the photon is its energy (hf) divided by the speed of light in themedium. That is, p=hfn/c, where f is the frequency, n is the refractiveindex of the sphere, and c is the speed of light in vacuum.Consequently, the angular momentum may be expressed as:

$\begin{matrix}{L = {{\frac{hfna}{c}\cos\frac{\pi}{q}} = {\frac{hna}{\lambda}\cos\frac{\pi}{q}}}} & (2)\end{matrix}$where λ is the wavelength in vacuum.

Since the resonance mode has a constant angular momentum, equation (2)can be used to estimate the effect that various perturbations have onthe resonance wavelength. For example, to reiterate, as was illustratedin FIG. 4, if the size (or shape, or refractive index) of the particle100/100′ changes, the resonances shift in frequency. In the case of asphere, as its radius increases, the resonance occurs at a longerwavelength. This shift can be expressed as:

$\begin{matrix}{\frac{\Delta\; a}{a} = \frac{\Delta\;\lambda}{\lambda}} & (1)\end{matrix}$

The sensitivity of this measurement technique can be estimated asfollows. If it is assumed that the linewidth (δλ≅10⁻⁸λ), then thesmallest “measurable” size change is |Δa|_(min)=10⁻⁸a. Assuming a sphereradius (a) on the order of 10 μm, |Δa|_(min)=10⁻¹³ m. This is muchsmaller than the size of an atom.

Unfortunately, the resonance of photon(s) orbiting within a microsphereis fairly sensitive to changes in temperature. To estimate the resonanceshift due to temperature change, both the radius and refractive index(n) of the microsphere are permitted to vary. Based on equation (2), thefractional shift in wavelength may be expressed as:

$\begin{matrix}{\frac{\Delta\;\lambda}{\lambda} = {\frac{\Delta\; a}{a} + \frac{\Delta\; n}{n}}} & (3)\end{matrix}$In most amorphous optical materials, both the size and the refractiveindex will change approximately linearly with temperature at near roomtemperature. Thus, there is a need for improving the foregoing techniqueof detecting and/or measuring a substance based on a resonance shift ofphotons orbiting within a microsphere, by making it insensitive or lesssensitive to changes in temperature. Indeed, it would be useful to makethe foregoing technique insensitive or less sensitive to changes otherthan changes in the amount or concentration of the substance beingdetected or measured.

Other challenges to using the foregoing technique include (i) connectingthe microsphere to the optical fiber to ensure adequate mechanicalreliability and adequate optical coupling, and (ii) attaching receptorsto the microsphere.

§ 2. SUMMARY OF THE INVENTION

The present invention may provide a detection and/or measurementtechnique based on a resonance shift in photons orbiting within amicrosphere. The present invention may do so by applying a light sourceto a sensor including a microsphere coupled with an optical carrier,detecting light at the other end of the optical carrier, and determiningadsorption of a material onto the microsphere based on the detectedlight.

The present invention may also provide improved techniques for attachinga microsphere to optical fiber. The present invention may do so byeroding cladding from the optical fiber and using a siloxane network tobridge a silica fiber and a silica microsphere, or using amide and/orother bonds to bridge a silica microsphere and a silica fiber, orattaching carboxulic acid to the eroded fiber with a copolymer of methylmethacylate and acrylic acid in solution, and bridging the twocarboxylic groups.

The present invention may also provide improved techniques for attachingreceptors to a microsphere. The present invention may do so bycovalently bonding complementary oligonucleotides to the surface of amicrosphere, covalently attaching an antibody to the surface of amicrosphere, or immobilizing an enzyme on the microsphere.

Finally, the present invention may provide an improved detection and/ormeasurement technique, based on a resonance shift in photons orbitingwithin a microsphere, that is insensitive or less sensitive to changesin temperature or other factors unrelated to the presence orconcentration of the substance being detected or measured. The presentinvention may do so by providing a sensor with multiple microspheres anddistinguishing resonance shifts due to common mode noise from resonanceshifts due to the adsorption of a substance onto at least one of themicrospheres.

§ 3. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross sectional view of internal reflections in amicrosphere.

FIG. 2 illustrates the evanescent coupling of an optical fiber and amicrosphere.

FIG. 3 illustrates transmission dips detected in light passing throughan optical fiber evanescently coupled with a microsphere, such as thatillustrated in FIG. 2.

FIG. 4 illustrates the shift in resonance of photon(s) orbiting within asphere as the size of the sphere changes.

FIG. 5 illustrates operations that may be performed by a system fordetecting and/or measuring a substance based on a resonance shift ofphotons orbiting within a microsphere.

FIG. 6 illustrates operations that may be performed when fabricating asensor head for use in a system, such as that illustrated in FIG. 5, fordetecting and/or measuring a substance based on a resonance shift ofphotons orbiting within a microsphere.

FIG. 7 illustrates an exemplary system for detecting and/or measuring asubstance based on a resonance shift of photons orbiting within amicrosphere.

FIG. 8 illustrates a single-sphere sensor head that may be used in asystem for detecting and/or measuring a substance based on a resonanceshift of photons orbiting within a microsphere.

FIG. 9 illustrates an exemplary single-sphere sensor.

FIG. 10 illustrates a multiple-sphere sensor head that may be used in asystem for detecting and/or measuring a substance based on a resonanceshift of photons orbiting within a microsphere.

FIG. 11 illustrates a multiple-sphere sensor in which one of threereceptors is modified with a receptor A to attract a ligand A′.

FIGS. 12A and 12B illustrate the frequency spectra of light detectedthrough the sensors of FIGS. 11 and 13, respectively.

FIG. 13 illustrates the multiple-sphere sensor head of FIG. 11 placedinto a solution including substance A′.

FIG. 14 is a flow diagram of an exemplary measurement method for usewith a single-sphere sensing head.

FIG. 15 is a flow diagram of an exemplary measurement method for usewith a multiple-sphere sensing head.

FIG. 16 is a cross-sectional end view of a microsphere coupled with anoptical fiber.

FIGS. 17A and 17B illustrate symmetric and asymmetric contact,respectively, between a microsphere and an optical fiber.

FIG. 18 is a resonance frequency spectrum of detected light that haspassed through a sensing head.

FIG. 19 illustrates an experimental system used to observe the resonancefrequencies of a microsphere connected with an optical fiber.

FIG. 20 is a cross-sectional end view of a microsphere coupled onto acylindrically eroded fiber.

FIGS. 21A and 21B illustrate a silica fiber with cladding andcylindrically eroded cladding, respectively.

§ 4. DETAILED DESCRIPTION OF THE INVENTION

The present invention involves novel methods and apparatus for detectingand/or measuring a substance based on a shift in resonance of photon(s)orbiting within a microsphere. The following description is presented toenable one skilled in the art to make and use the invention, and isprovided in the context of particular embodiments and methods. Variousmodifications to the disclosed embodiments and methods will be apparentto those skilled in the art, and the general principles set forth belowmay be applied to other embodiments, methods and applications. Thus, thepresent invention is not intended to be limited to the embodiments andmethods shown and the inventors regard their invention as the followingdisclosed methods, apparatus and materials and any other patentablesubject matter to the extent that they are patentable.

§ 4.1 Exemplary Applications

The present invention may be used in detecting and/or measuringsubstances, such as chemical or biological substances for example. Theparticular substance to be detected and/or measured may affect thedesign (e.g., size, material, etc.) of the microsphere, as well as thechoice of receptors. Exemplary sensors, used to detect RNA, DNA strands,antigens, bacteria, and other biological substances, as well as chemicalsubstances, are described in § 4.4.2 below.

§ 4.2 Functions that May be Performed

The present invention may function to detect and/or measure a substancebased on a resonance shift of photons orbiting within a microsphere. Thepresent invention may also function to attach a microsphere to opticalfiber. The present invention may also function to attach receptors to amicrosphere. Finally, the present invention may function to improvedetection and/or measurement, based on a resonance shift of photonsorbiting within a microsphere, by eliminating or reducing sensitivity tochanges in temperature or other factors unrelated to the presence orconcentration of the substance being detected or measured.

§ 4.3 Exemplary Operations

FIG. 5 is a bubble chart illustrating operations that may be performedin detecting and/or measuring a substance in accordance with the presentinvention. A light sourcing operation 520 may emit a light, under thecontrol of a light source control operation 510, into (or through) asensor 530. A detection operation 540 may detect light from the sensor530. Certain detected properties of the light may then be provided todetection and/or measurement operation(s) 550. Exemplary methods andapparatus that may be used to effect these various operations aredescribed in § 4.4.1 below.

Certain aspects of the present invention concern the fabrication of asensor to be used in a system such as that illustrated in FIG. 5. FIG. 6is a bubble chart illustrating operations that may be performing infabricating a sensor in accordance with the present invention.Basically, the fabrication of a sensor in accordance with the presentinvention may include two operations—attaching 610 the receptor(s) tothe microsphere(s), and coupling 620 the microsphere(s) to the opticalfiber. Exemplary methods, apparatus and materials that may be used toeffect these various operations are described in § 4.4.2 below.

§ 4.4 Exemplary Methods and Apparatus for Performing the ExemplaryOperations

Exemplary methods and apparatus that may be used to perform operationsrelated to detecting and/or measuring a substance are described in §4.4.1 below. Then, exemplary methods, apparatus and compositions ofmatter that may be used to perform operations related to fabricatingsensors are described in § 4.4.2 below.

§ 4.4.1 Exemplary Methods and Apparatus for Performing OperationsRelated to Detecting and/or Measuring Substances

In the following, exemplary apparatus for detecting and/or measuringsubstances are described in § 4.4.1.1, while exemplary methods aredescribed in § 4.4.1.2.

§ 4.4.1.1 Exemplary Apparatus for Detecting and/or Measuring Substances

FIG. 7 is a block diagram of a system 700 that may be used to effect theoperations of FIG. 5. A computing device, such as a personal computerfor example, may function to (a) control a tunable laser 720, and (b) todetermine the existence or amount of a substance based on the output ofa photodetector 740.

§ 4.4.1.1.1 Exemplary Light Source

As can be appreciated from the foregoing, the tunable laser 720 mayeffect the light sourcing operation(s) 520. An exemplary tunable laser720 is the model 2010A, available from Newport Corporation of Irvine,Calif., which permits scanning the CW laser with an external cavity. Thetuning range of the exemplary laser 720 may be ±4 nm with the centerwavelength at 635 nm. The linewidth of the exemplary laser 720 may beless than 1 kHz, with a resolution on the order of 10⁻¹¹.

§ 4.4.1.1.2 Exemplary Detector

As can also be appreciated from the foregoing, the detector 740 mayeffect the light detecting operation(s) 540. An exemplary detector 740is the model PDA55 broadband photodiode detector available fromThorlabs, Inc. of Newton, N.J. The output of the detector may bedigitized by an analog-to-digital converter, such as the 16-bit, 200 kHzmodel PCI-6034E from National Instruments Corporation of Austin, Tex.,in the personal computer.

§ 4.4.1.1.3 Exemplary Sensing Heads

Although not shown, standard optical fiber connectors may be provided tofacilitate the exchange of sensing heads 530.

The sensing head 530 may have a number of possible configurations, twoof which are described in detail below. The first sensing headconfiguration is referred to as a single-sphere sensing head. The secondsensing head configuration is referred to as a multiple-sphere sensinghead. In either case, the radius of the microsphere(s) preferably rangesfrom about 2 μm to about 1 mm, and more preferably is from about 10 μmto about 100 μm.

§ 4.4.1.1.3.1 Single-Sphere Sensing Head

As shown in FIG. 8, an exemplary single-sphere sensing head 800 mayinclude a microsphere 810 positioned at the end of a pair of opticalfibers 820/830. One of the optical fibers 820 is optically coupled witha light source, while the other is optically coupled with aphotodetector. For example, FIG. 9 is a cross-section of a possibledesign for a single-sphere sensing head 900 including a microsphere 900positioned at the end of a pair of optical fibers 920/930. Lightdirected through optical fiber 920 toward the microsphere 910 may reachthe beveled surface of the fiber at an angle θ, such as the one greaterthan the critical angle θ_(c) (sin θ_(c)=n_(sphere)/n_(core), wheren_(sphere) and n_(core) are the refractive indices of the sphere and thefiber core, respectively). The evanescent field just outside the opticalfiber 920 couples to the evanescent field of a particular resonant modeof the microsphere 910. The other optical fiber 930 senses thisevanescent field around the microsphere 910. Consequently, peaks can beobserved in the outgoing optical fiber 930 at the resonant frequencies.An exemplary single-sphere sensing head having the general features asthat 900 illustrated in FIG. 9 may be fabricated in the manner describedin § 4.4.2 below.

§ 4.4.1.1.3.2 Multiple-Sphere Sensing Head

A multiple-sphere sensing head will now be described. First, however,the challenges that led to the multiple-sphere sensing head areintroduced. Recall that the resonance characteristics of a microsphereare based on (i) the size of the microsphere, (ii) the shape of themicrosphere, (iii) the refractive indices of the microsphere and thesurrounding medium, and (iv) the adsorption of the microsphere. Some ofthese factors will be influenced by the local temperature, the stress onthe microsphere, and the concentration or the presence of the substanceto be measured in the surrounding medium (e.g., fluid). Recall also thatthe sensitivity of the resonance frequencies to changes in temperatureis relatively high. Indeed, the resonance frequencies of the microspheremay be extremely susceptible to environmental disturbances. For example,a slight drift in temperature, or pressure of the surrounding medium, ora change in a solvent composition (and consequently, its refractiveindex) in which the microsphere is placed, may cause a large change inthe resonance characteristics, perhaps exceeding the change caused bythe adsorption of the substance or the presence of the substance inclose proximity of the microsphere surface. A multiple-sphere sensinghead will mitigate or eliminate these problems.

As shown in FIG. 10, an exemplary multiple-sphere sensing head 1000 mayinclude at least two microspheres 1010 coupled with an optical fiber1020. One end of the optical fiber 1020 is optically coupled with alight source, while the other end is optically coupled with aphotodetector. In one embodiment, the surface of each microsphere 1010is modified with a receptor to interact with a specific ligand, thoughat least one of the microspheres may 1010 remain unmodified. In such anembodiment, changes due to the environmental disturbances affect theresonance characteristics for all of the microspheres 1010 in the sameway. This affect on the resonance characteristics of the microspheres1010 can be characterized as “common-mode noise”. On the other hand, theadsorption of a specific ligand will affect only the microsphere(s) 1010having the associated receptor. The common-mode noise can be removedfrom the signal using wavelength screening and spectrum interpretation.An example of this is illustrated in FIGS. 11, 12A, 12B and 13.

FIG. 11 illustrates a multiple-sphere sensing head 1000′ including anoptical fiber 1020′ and three microspheres 1010 a′, 1010 b′ and 1010 c′.The microsphere 1010 a′ is modified with a receptor for ligand A. FIG.12A illustrates the dips in the frequency spectrum associated with aresonance frequency associated with each of the microspheres 1010 a′,1010 b′ and 1010 c′. Notice that the distinct dips can be used todistinguish the microspheres. FIG. 13 illustrates the multiple-spheresensing head 1000′ immersed in a solution 1100 containing substance A.Notice from FIG. 12B that all of the frequencies of all of the dips,associated with the resonant frequencies of the microspheres 1010 a″,1010 b′ and 1010 c′ have all shifted to some extent due to common-modenoise, but the frequency of the dip associated with the resonantfrequency of microsphere 1010 a″ will have also shifted (in the same oropposite direction as the shift due to the common-mode noise) due to thechange in the size of microsphere 1010 a″ resulting from the adsorptionof substance A. As can be seen in FIGS. 12A and 12B, the shift due toadsorption can be distinguished from that due to the noise.

§ 4.4.1.2 Exemplary Methods for Detecting and/or Measuring Substances

Exemplary methods for detecting and/or measuring substances using asingle-sphere sensing head and a multiple-sphere sensing head, aredescribed with reference to FIGS. 14 and 15, respectively.

FIG. 14 is a flow diagram of an exemplary method 520′/540′/550′ that maybe used to effect light sourcing operations 520, light detectionoperations 540, and substance detection and/or measurement operations550 used with a single-sphere sensor head. The order in which the actsare performed is not intended to be limited by the order shown in FIG.14. As shown in block 1405, a light source is applied. The light sourcemay be a tunable laser, for example, and may be applied to a first fiberof single-sphere head sensor (Recall, e.g., 920 of FIG. 9.) or a firstend of a fiber having an attached micro-sphere. As indicated by block1410, light is detected. The light may be detected by a broadband,photodiode detector, for example, which may be coupled with a secondfiber of a single-sphere head sensor (Recall, e.g., 930 of FIG. 9.), orwith a second end of a fiber having an attached micro-sphere. Theresonant frequencies, seen as dips (Recall, e.g., FIG. 3.), may berecorded, as indicated by block 1415. Then, as indicated by block 1420,the sensing head is placed in the environment (e.g., a solution) whichmay include the substance to be detected and/or measured. As was thecase with blocks 1405 and 1410, a light source is (or continues to be)supplied and the resulting light is detected as indicated by acts 1425and 1430, respectively. As was the case with block 1415, the resonantfrequencies, seen as dips, may be recorded, as indicated by block 1435.The change in resonant frequency (or the change in the associatedwavelength) is determined, as indicated in block 1440. This may simplybe a matter of determining the differences between the dips before andafter the sensing head is placed in the environment (e.g., solution)which may include the substance to be detected and/or measured. Finally,as indicated by block 1445, the determined change in resonant frequency(or determined change in the wavelength) are converted to a detection ormeasurement of the substance. As indicated by the bracket adjacent toblocks 1425 through 1445, these acts may be repeated to determineadsorption on other microspheres, for example, which may be converted toa concentration of another substance. The method may be left via RETURNnode 1450.

Recall from § 4.4.1.1.3.2 above that by using a multiple-sphere sensinghead, common-mode noise can be removed from the signal using wavelengthscreening and spectrum interpretation. That is, since the frequencies ofall of the dips, associated with the resonant frequencies of themicrospheres, all shift to some extent due to common-mode noise, but thefrequency of the dip associated with the resonant frequency of amicrosphere with receptors will also shift (in the same or oppositedirection as the shift due to the common-mode noise) due to the changein the size of that microsphere resulting from the adsorption of asubstance, the shift due to adsorption can be distinguished from thatdue to the noise.

FIG. 15 is a flow diagram of an exemplary method 520″/540″/550″ that maybe used to effect light sourcing operations 520, light detectionoperations 540, and substance detection and/or measurement operations550 used with a multiple-sphere sensor head. The order in which the actsare performed is not intended to be limited by the order shown in FIG.15. As shown in block 1505, a light source is applied. The light sourcemay be a tunable laser, for example, and may be applied to a first endof a fiber having attached micro-spheres (Recall, e.g., FIG. 11.). Asindicated by block 1510, light is detected. The light may be detected bya broadband photodiode detector, for example, which may be coupled witha second end of the fiber having the attached micro-spheres. Theresonant frequencies, seen as dips (Recall, e.g., FIG. 12A.), may berecorded, as indicated by block 1515. These resonant frequencies areassociated with the various microspheres as indicated by block 1517.This may be done by monitoring a transmission spectrum through thefiber. For example, recall from equation (1) that Δλ is proportional toΔa. Resonance from two microspheres differing by 10⁻⁷a should be easilydistinguishable. Given that the standard deviation in “a” for emulsionpolymerization of a polystyrene microsphere is >10⁻²a, the resonancefrequency of each microsphere, among ˜1000 of microspheres, should beeasily distinguished from those resonance frequencies of the othermicrospheres. Then, as indicated by block 1520, the sensing head isplaced in the environment (e.g., a solution) which may include thesubstance to be detected and/or measured. As was the case with blocks1505 and 1510, a light source is (or continues to be) supplied and theresulting light is detected as indicated by acts 1525 and 1530,respectively. As was the case with block 1515, the resonant frequencies,seen as dips, may be recorded, as indicated by block 1535. As was thecase with block 1517, the resonant frequencies are associated with themicrospheres, as indicated by block 1536.

Common-mode noise may then be determined, as indicated by block 1537.(Recall, e.g., FIGS. 12A and 12B.) The total change in resonantfrequency (or the total change in wavelength) for the microsphere(s)provided with the receptor(s) is then determined as indicated by block1538. Then, as indicated by block 1540, the amount of change in resonantfrequency (or wavelength) due to common-mode noise is removed from thetotal change in resonant frequency (or wavelength) to obtain the part ofthe change attributable to the presence of the substance being detectedand/or measured. Finally, as indicated by block 1545, the part of thedetermined change in resonant frequency (or determined change in thewavelength) attributable to the presence of the substance is convertedto a detection or measurement of the substance. As indicated by thebracket adjacent to blocks 1525 through 1545, these acts may be repeatedto determine a rate of adsorption, for example, which may be convertedto a concentration of the substance. The method may be left via RETURNnode 1550.

§ 4.4.2 Exemplary Methods, Apparatus and Compositions of Matter forPerforming Operations Related to Fabricating Sensors

Recall from FIG. 5 that the system 500 uses a sensor 530. Recall fromFIGS. 9 and 11 that a sensor may be characterized as a single-spheresensing head or a multiple-sphere sensing head. In each case, assummarized in FIG. 6, the fabrication of a sensor head involves twobasic operations—and coupling the microsphere(s) and optical fiber(s),and attaching receptor(s) to a microsphere(s). Methods and apparatusthat may be used to perform these operations, as well as the resultingsensors, are now described. Note that the methods, apparatus, andcomponents used will often depend upon the ultimate application of thesensing head.

§ 4.4.2.1 Exemplary Materials for the Fiber and Microspheres

An inorganic glass, such as silica, or an amorphous polymer, such aspoly(methyl methacrylate) (“PMMA”), are suitable materials for theoptical fiber. Other known materials for optical fiber may be used.

The microsphere(s) can be any transparent material, such as silica,suffire, BK7, polystyrene, PMMA, polycarbonate, poly(ethyleneterephthalate), etc. Spheres of different diameters are commerciallyavailable (such as from PolySciences, Inc., of Warrington, Pa.). Forapplications such as in vitro and in vivo measurement of chemicals inthe blood vessel or body fluid, PMMA may be an appropriate material.Other polymers may also be suitable microsphere materials since they areinert in biological materials. Many polymers are also advantageouslystable in acidic and basic environments. In such applications, thesurface of the PMMA spheres may be modified to make them biocompatibleand hypoallergenic. (See, e.g., Lasting Correction of Skin Defects andWrinkles, http://www.canderm.com/artecoll/tech.html. With appropriatesurface modifications, inorganic glasses may be also renderedbiocompatible.

The microsphere(s) and the fiber may be made of the same material,though this is not necessary. However, it is preferable to keep therefractive indices of the microsphere(s) and fiber close to each otherto promote phase matching.

§ 4.4.2.2 Exemplary Methods for Coupling the Fiber and Microsphere(s)

In the following, the term “connection” will be used to generally referto all (e.g., mechanical, optical, electro-magnetic, etc.) interactionsbetween a microsphere and a fiber. The term “coupling” will refer to theevanescent connection of a microsphere and a fiber, while the term“bridge” will refer, without loss of generality, to the mechanicalconnection of a microsphere and a fiber. Some theory related todesirable connection characteristics is first introduced in § 4.4.2.2.1.Then the affects of symmetry, distance, and mechanical bridging aredescribed in §§ 4.4.2.2.2 through 4.4.2.2.4 below. Finally, someexemplary methods and compositions of matter for attaching the fiber andmicrosphere(s) are described in § 4.4.2.2.5.

§ 4.4.2.2.1 Optical Coupling Via Evanescent Field

By overlapping the evanescent field that surrounds a microsphere, andtypically extends for a characteristic length of about 0.1 μm from thesurface, with the evanescent field from the core of an optical fiber,(optical) coupling can be achieved. (See, e.g., the article A.Serpenguzel, S. Arnold, G. Griffel, J. A. Lock, Efficient Coupling ofGuided Waves to Microsphere Resonances Using an Optical Fiber, J. Opt.Soc. B, 14, 790 (1997).) A cross-sectional end view of a basichalf-coupler 1600 is illustrated in FIG. 16. In the half-coupler 1600,light is directed through the optical fiber 1610 (into the page). Thefiber cladding 1612 is eroded to expose the evanescent field justoutside the core 1614. The eroded fiber 1610 is pressed against themicrosphere 1620.

§ 4.4.2.2.2 Effects of Symmetry of a Microsphere-to-Fiber Connection

It is desirable to provide a symmetric connection between the erodedfiber 1610 and the microsphere 1620, such as shown in FIG. 17A. If thecenter of the microsphere 1620 is not located above the center of thefiber core 1614, such as illustrated in FIG. 17B, coupling may besubstantially attenuated. This was observed in the resonance spectrum,shown in FIG. 18, of an experimental system illustrated in FIG. 19.

More specifically, in the experimental system 1900 of FIG. 19, a fiber1910 was epoxied to a Lucite block 1915. The combination was sanded andpolished to eliminate most of the cladding 1912 from one side of thefiber 1910. Light from a tunable dye laser 1950 was coupled into thefiber with a polystyrene microsphere 1920 having a radius ≅15 μm. Themicrosphere 1920 was immersed in water. In one experiment, no scattering(i.e., radiation leakage) was observed from the microsphere 1920 untilthe wavelength was tuned to resonance, although scratches on thepolished surface caused a small amount of background scattering. Asindicated on the detected spectrum 1800 of FIG. 18, three orders ofresonance occurred, the narrowest of which was essentially the same asthat of the dye laser resolution (e.g., 0.025 nm). Note that the nearlyperiodic repeating resonance of a given order are associated with themode number. Since the measured spectrum is a convolution of theintrinsic resonance line and the laser line, the actual resonance widthis considerably narrower than measured. Wave theory can be used topredict the position of all of the resonance in the spectrum 1800 usingjust one adjustable parameter “a”. Note, however, that near thebeginning of the resonance spectrum 1800, the first-order modes are notpresent.

Such undesirable asymmetric contact may occur if the microsphere (a) isimproperly positioned, and/or (b) moves (e.g., rolls) out of position.To secure the symmetric coupling between the evanescent fields of themicrosphere and the fiber by ensuring symmetric contact such as thatillustrated in FIG. 17A, a polymer microsphere may be attached,covalently, with the eroded fiber. Details of the covalent attachmentare given in 4.4.2.2.5.

§ 4.4.2.2.3 Effects of Separation Distance of a Microsphere-to-FiberConnection

In addition to the desirability of symmetric coupling, the distancebetween the microsphere and fiber core will also affect performance.More specifically, if the microsphere and fiber core are too far apart,the coupling of their respective evanescent fields may be insufficient.If, on the other hand, the microsphere and fiber core are too close, thepresences of the fiber's evanescent field may change the boundarycondition of the microsphere, thereby undermining the inherently highquality factor (Q) of the resonance. However, the inventors have foundthat permitting the microsphere and fiber core to contact one another isacceptable in some applications.

§ 4.4.2.2.4 Effects of the Bridging of a Microsphere-to-Fiber Connection

Further, the bridge physically coupling the microsphere(s) and the fibershould be mechanically strong and durable. However, the coupling shouldminimize the perturbation to the resonating state of the photon(s) inthe microsphere. Thus, for example, a bridge coupling each microspherewith the fiber should be small.

§ 4.4.2.2.5 Exemplary Methods and Compositions of Matter for Attachingthe Fiber and Microsphere(s)

FIG. 20 is a cross-sectional side view of a sensing head (section) 2000including an attached microsphere 2020 and fiber 2010. To locate themicrosphere 2020 symmetrically, with respect to the fiber core 2014, thefiber 2010 was eroded on all sides (referred to as “cylindricalerosion”). Such cylindrical erosion of the cladding 2012 can be effectedby etching the fiber 2010 at a desired region or regions with ahydrofluoric acid solution or a base solution, thereby exposing theevanescent field of the fiber core 2014. In one exemplary fabricationmethod, the etching was terminated when the amplitude of a lasertransmitting through the fiber showed a hint of decrease. As illustratedin FIGS. 21A and 21B, a silica fiber with cladding had an initial totaldiameter of ˜125 μm, before being eroded to a diameter of ˜6 μm. Thefinal diameter can be controlled by changing the concentration of theacid or the base and the etching time.

Plastic fiber may be eroded by immersing it into a solvent thatdissolves the cladding. For example, a PMMA fiber with a fluoropolymercladding (available from Mitsubishi Rayon and Toray) can be eroded in asolution of hexafluoroisopropanol.

Other methods for exposing evanescent field of the core of the opticalfiber will be apparent to those skilled in the art. Now, exemplarymethods and compositions of matter for attaching a microsphere to afiber are described. Generally though, the microspheres may be connectedto the eroded fiber with techniques used by biochemists for attachingmicrospheres to microscope slides (See, e.g., F. J. Steemers, J. A.Ferguson, D. R. Walt, Screening Unlabeled DNA targets with RandomlyOrdered Fiber Optic Gene Arrays, Natur. Biotech. 18, 91 (2000).), or byother silanization methods (See, e.g., The Colloid Chemistry of Silica,H. E. Bergna, ed. Adv. Chem. Ser. 234, Amer. Chem. Soc. (1994); E. P.Plueddemann, Silane Coupling Agents Kluwer (1990).).

In a first example, a siloxane network may be used to bridge a silicafiber and a silica microsphere. More specifically, a tiny amount (e.g.,˜Pico liter) of tetramethozysilane or dimethyldimethoxysilane may beapplied (e.g., dropped) into a space between a microsphere and thefiber, followed by dehydration and baking in an oven. (See, e.g., E. P.Plueddemann, Silane Coupling Agents Kluwer (1990).) The resultantfiber-sphere pair is chemically identical to bare silica.

In a second example, amide and other bonds may be used to bridge asilica microsphere and a silica fiber. More specifically, surfacesilanols on the microsphere and fiber can be converted to primaryamines. Consequently, the two amines will be bonded by acid anhydride ordialdehyde. Silica surface has a high density of reactive silanols(˜0.05 Å⁻²), or can at least be modified to have silanols at highdensity by washing in hydrochloric acid and rinsing followed by heating.Amino silanation will be accomplished by reacting silanols with aminosilanation agencies such as aminopropyl trimethoxysilane. The silanationmethods have been widely used to make glass fiber compatible to aplastic matrix to prepare fiber-reinforced plastic. (See, e.g., thearticle E. P. Plueddemann, Silane Coupling Agents Kluwer (1990).)Bridging two amines with acid anhydride such as succinic anhydride ordialdehyde such as glutaraldehyde is widely used in biochemistry. (See,e.g., the article J. McCafferty, H. R. Hoogenboom, D. J. Chiswell Ed.,Antibody Engineering, IRL Press (1996).)

Instead of amine modification, the silica surface can be modified withcarboxylic acid (by aminopropyl modification followed by reaction withsuccinic anhydride) and bridge two acids with carbodiimide.

The two foregoing methods form similar functional groups on both thefiber and microsphere. The inventors believe that one of thesefunctional groups can be modified with amine and the other of thesefunctional groups can be modified with carboxyl, so that the contactpoints can convert to amide bonds.

Amide bond formation is advantageous in that (i) the bonds are formedonly where the sphere and fiber are in contact, and (ii) the resultantmicrosphere-fiber complex retains reactive surface moieties for furtherbiochemical and biological functionalization.

In a third example, a plastic fiber is connected with a plasticmicrosphere. PMMA spheres having a carboxylated surface are commerciallyavailable, in various diameters, from PolySciences Inc. of Warrington,Pa. However, the PMMA core of optical fiber does not have a carboxylatedsurface (not functionalized). Carboxylic acid may be attached to theoptical fiber core by coating the eroded fiber with a copolymer ofmethyl methacrylate and acrylic acid in solution, followed by annealing.Thereafter, bridging the two carboxylic groups can be done in the samemanner as described above for bridging silica.

A tiny amount of silanization agent and a bridging agent may be provided(e.g., dropped), for example with a Pico liter jet (See, e.g., thearticle S. Arnold, L. M. Folan, A Fluorescence Spectrometer for a SingleElectrodynamically Levitated Microparticle, Rev. Sci. Inst. 57, 2250(1986).) onto the microsphere-fiber core contact.

Referring back to FIG. 9, a slightly spheroidal fused silica bead can beformed at the end of silica fibers by melting it with a microtorch.(See, e.g., the article V. B. Braginsky, M. L. Gorodetsky, V. S.Ilchenko, Quality-Factor and Nonlinear Properties of OpticalWhispering-Galley Modes, Phys. Lett. A, 137, 393 (1989).) In oneexperiment, a quality factor (Q) of 3×10⁷ was maintained for such asensing head 900.

§ 4.4.2.3 Providing Receptor on Microsphere(s)

Recall that the surface of a microsphere used to detect a substance willbe modified with a receptor to interact with that substance (a specificligand). Naturally, the receptor used will depend upon the particularapplication for which the sensor head is to be used. Various exemplarymicrosphere-receptor combinations are described below.

In a first example, the microsphere is modified to adsorb a specificligand. First, surface silanols are functionalized onto the microsphere.Then, a secondary modification is used to attach the biochemicalsubstance complementary to the specific ligand to be detected. Thecoupling utilizes reactions between two amines, two acids, or amine andacid. Amino groups naturally present in proteins can be coupled directlyto the surface carboxyl or with glutaraldehyde to the surface amine.Attaching glucose oxidase to the microsphere surface will allow thedetection of glucose for instance.

In a second example, it is desired to detect RNA and single-stranded DNAfragments. In such an application, the microspheres may be provided withcomplementary oligonucleotides covalently bonded to their surface.Covalent bonding will be furnished by coupling nucleotides (withalkylamine extended on the 5′ end) with the surface amine usingglutaraldehyde. When used in a system such as that of FIG. 5, suchmicrospheres would experience a shift in their optical resonance whenhybridized with complementary strands. Adsorbates which are notcomplementary may be washed away. By preparing a plurality ofmicrospheres with different oligonucleotide modifications and arrangingthem along an optical fiber to provide a multiple-sphere sensing head,it is possible to identify the base sequence in a given DNA sample. Sucha sensing head could be calibrated by adding a known complementaryoligonucleotide and observing the corresponding dip in the spectrumshifts. The bound oligonucleotides can then be removed, for instance, bysubsequent heat denaturing. In this way, single-molecule detection, withor without fluorescence labeling, is possible.

In a third example, it is desired to detect the presence of an antigensuch as carcinoembryonic (CEA) antigen and HSA (human serum albumin),etc. . In such an application, an antibody or antibodies such asanti-CEA and anti-HSA, etc. may be covalently attached to the surface ofa microsphere. Conversely, presence of antibody may be detected byattaching its antigen molecules onto the surface.

In a fourth example, it is desired to detect the presence of a substrateand/or inhibitor. In such an application, the surface of a microspheremay be provided with an enzyme. For example, if glucose oxidase isimmobilized onto the surface of a microsphere, such a sensing head couldbe used in a system, such as that illustrated in FIG. 5, for detectingthe concentration of glucose. In another example, lipase immobilizedonto the surface of a microsphere will be able to detect the presence ofmagnesium ion, an inhibitor of the enzyme.

§ 4.5 Conclusions

It is clear from the foregoing that the present invention provides asmall, highly-sensitive with a high quality factor (Q), sensing head andsystem for detecting and/or measuring various substances, such asbiochemical substances. The resolution and dynamic range of theresonance far exceeds those of existing detection schemes. Indeed, thehigh quality factor (Q) detection enables unprecedented opportunitiesfor microscale sensing. Common-mode noise can be determined and removedby using a multiple-sphere sensing head. By modifying the surface of themicrosphere with biological receptors, the sensing head will interactwith specific biological ligands, allowing detection of the presence orthe concentration of the ligands.

1. For use in a system including a light source, and a light detector,for determining the presence or concentration of a substance in amedium, a sensor comprising: a) at least one optical fiber; b) aplurality of microspheres, each of the plurality of microspheres beingcoupled with the optical fiber, and at least one of the microsphereshaving a surface including receptors for the substance, wherein, whenlight is applied to the optical fiber, a resonance within each of themicrospheres is excited, wherein, if the substance adsorbs to thereceptors on a surface of one of the microspheres, a shift in theresonance occurs, wherein a presence or concentration of the substancecan be determined based on the shift in resonance, wherein a resonancefrequency of each of the plurality of microspheres can be distinguishedfrom resonance frequencies of the other of the plurality ofmicrospheres, and wherein a quality factor of the resonance excitedwithin at least one of the microspheres by the light applied to theoptical fiber is at least 10⁵.
 2. The sensor of claim 1 wherein thesubstance is one of RNA and single stranded DNA, and wherein thereceptors are complementary oligonucleotides.
 3. The sensor of claim 2wherein the complementary oligonucleotides are covalently bonded to thesurface of at least one of the plurality of microspheres.
 4. The sensorof claim 1 wherein the substance is an antigen, and wherein thereceptors are antibodies.
 5. The sensor of claim 4 wherein theantibodies are covalently bonded to the surface of at least one of theplurality of microspheres.
 6. The sensor of claim 1 wherein thesubstance is one of a substrate and an inhibitor, and wherein thereceptors are enzymes.
 7. The sensor of claim 1 wherein the substance isglucose, and wherein the receptors are glucose oxidase.
 8. The sensor ofclaim 1 wherein the optical fiber is an inorganic glass.
 9. The sensorof claim 8 wherein the optical fiber is silica.
 10. The sensor of claim1 wherein the optical fiber is an amorphous polymer.
 11. The sensor ofclaim 10 wherein the optical fiber is polymethyl methacrylate.
 12. Thesensor of claim 1 wherein the optical fiber is silica, wherein at leastone of the plurality of microspheres is silica; and wherein the at leastone microsphere and optical fiber are bridged by a siloxane network. 13.The sensor of claim 1 wherein the optical fiber is silica, wherein atleast one of the plurality of microspheres is silica, and wherein the atleast one microsphere and optical fiber are bridged by amine and otherbonds.
 14. The sensor of claim 13 wherein the other bonds bridge theamines, and are selected from a group consisting of acid anhydride,succinic anhydride, dialdehyde, and glutaraldehyde.
 15. The sensor ofclaim 1 wherein the optical fiber is polymethyl methacrylate, wherein atleast one of the plurality of microspheres is polymethyl methacrylate,and wherein the at least one microsphere and optical fiber are bridgedby a siloxane network.
 16. The sensor of claim 1 wherein at least one ofthe plurality of microspheres has a radius on the order of 10 μm. 17.The sensor of claim 1 wherein at least one of the microspheres iscoupled with the optical fiber in such a way that there is an evanescentconnection of the at least microsphere and the optical fiber.
 18. Thesensor of claim 1 wherein at least one of the microspheres is coupledwith the optical fiber in such a way that an evanescent fieldsurrounding the at least one microsphere overlaps an evanescent fieldfrom a core of the optical fiber.
 19. The sensor of claim 1 wherein atleast one of the microspheres is coupled with the optical fiber in sucha way that an evanescent field surrounding the at least one microsphereoverlaps an evanescent field from a core of the optical fiber to providea symmetric coupling between the evanescent fields.
 20. The sensor ofclaim 1 wherein the resonance excited within the microsphere is aresonance of photons orbiting within the microsphere.
 21. For use in asystem including a light source, and a light detector, for determiningthe presence or concentration of a substance in a medium, a sensorcomprising: a) an optical fiber; b) a microsphere i) being coupled withthe optical fiber, ii) having a surface including receptors for thesubstance, wherein, when light is applied to the optical fiber, aresonance within the microsphere is excited, wherein, if the substanceadsorbs to the receptors on the microsphere surface, a shift in theresonance occurs, wherein a presence or concentration of the substancecan be determined based on the shift in resonance, and wherein a qualityfactor of the resonance excited within the microsphere by the lightapplied to the optical fiber is at least 10⁵.
 22. The sensor of claim 21wherein the substance is one of RNA and single stranded DNA, and whereinthe receptors are complementary oligonucleotides.
 23. The sensor ofclaim 21 wherein the substance is an antigen, and wherein the receptorsare antibodies.
 24. The sensor of claim 21 wherein the substance is oneof a substrate and an inhibitor, and wherein the receptors are enzymes.25. The sensor of claim 21 wherein the substance is glucose, and whereinthe receptors are glucose oxidase.
 26. The sensor of claim 21 wherein atleast one of the microspheres is coupled with the optical fiber in sucha way that an evanescent field surrounding the at least one microsphereoverlaps an evanescent field from a core of the optical fiber to providea symmetric coupling between the evanescent fields.
 27. The sensor ofclaim 21 wherein the resonance excited within the microsphere is aresonance of photons orbiting within the microsphere.
 28. For use in asystem including a light source, and a light detector, for determiningthe presence or concentration of a substance in a medium, a sensorcomprising: a) at least one optical fiber; b) at least one microsphere,the at least one microsphere i) being coupled with the optical fiber,ii) having a surface including receptors for the substance, wherein,when light is applied to the optical fiber, a resonance within themicrosphere is excited, wherein, if the substance adsorbs to thereceptors on the microsphere surface, a shift in the resonance occurs,wherein a presence or concentration of the substance can be determinedbased on the shift in resonance, and wherein the at least onemicrosphere is coupled with the optical fiber at a beveled surface ofthe optical fiber such that light directed through the optical fibertowards the at least one microsphere reaches the optical fiber at anangle greater than a critical angle, wherein the critical angle is thearcsine of the refractive index of the microsphere divided by therefractive index of the optical fiber.